Abstract Loss of homeostatic relationships with microbiota can result in inflammatory diseases such as the inflammatory bowel diseases (IBD). Maintaining homeostasis with microbiota is predicated on the ability of host cells to adjust their transcriptional programs in response to signals from microbiota. Intestinal epithelial cells (IECs) serve critical roles as a barrier against microbiota. IECs perform these roles by integrating external signals into various transcriptional programs which output appropriate physiologic responses. Hepatocyte nuclear factor 4 alpha (HNF4A) is a nuclear receptor transcription factor (TF) that is highly expressed in the vertebrate digestive tract. In IECs, HNF4A acts predominantly as a transcriptional activator regulating genes involved in IEC development, barrier function, metabolism, and nutrient absorption. Genetic variants at the HNF4A gene locus and HNF4A transcription factor binding motifs have been identified in GWAS for human IBD. Intestine specific knockout of Hnf4a in mice results in highly penetrant spontaneous intestinal inflammation. However, it is unknown whether this phenotype is due to intrinsic anti-inflammatory roles for HNF4A in IECs or barrier defects that result in activation of immune cells in the lamina propria. The overall objective of this project is to understand the role of HNF4A in maintaining homeostasis with microbiota in the intestine and in regulating microbiota-responsive enhancers in IECs. Our lab recently made the key discovery that HNF4A activity in the IEC genome is suppressed by microbiota in mice and zebrafish. Additionally we found that Hnf4a protects zebrafish from a microbiota-driven transcriptional shift which correlates with transcriptional shifts seen in human IBD. We also identified a subset of enhancers that are regulated by the microbiota in mouse IECs, which are also bound by HNF4A. However the role of HNF4A in maintaining intestinal homeostasis with microbiota by acting on these enhancers is unknown. I will test the central hypothesis that HNF4A promotes intestinal homeostasis in the presence of microbiota by mediating microbiota-induced alterations in enhancer activity across the IEC genome. In Specific Aim 1, I will derive a mouse strain lacking Hnf4a in IECs (Hnf4a?IEC) into germ free (GF) conditions to test the role of microbiota in the intestinal inflammation phenotype of this mouse model of IBD. Additionally, I will generate enteroid cultures from primary IECs of these mice to study the role of HNF4A in mediating IEC intrinsic responses to microbiota in vitro. In Specific Aim 2, I will define the role of HNF4A at microbiota responsive enhancers using GF and ex-GF conventionalized (CV) Hnf4a?IEC mice and wild-type controls. The outcomes of this work will provide innovative in vivo genetic evidence establishing the role of HNF4A as a mediator of IEC transcriptional programs protective against microbiota-driven intestinal inflammation. This research addresses a critical knowledge gap of the role of HNF4A in maintaining intestinal homeostasis with microbiota.